Zika Virus Infection during Pregnancy in Mice Causes Placental Damage and Fetal Demise.

Zika virus (ZIKV) infection in pregnant women causes intrauterine growth restriction, spontaneous abortion, and microcephaly. Here, we describe two mouse models of placental and fetal disease associated with in utero transmission of ZIKV. Female mice lacking type I interferon signaling (Ifnar1(-/-)) crossed to wild-type (WT) males produced heterozygous fetuses resembling the immune status of human fetuses. Maternal inoculation at embryonic day 6.5 (E6.5) or E7.5 resulted in fetal demise that was associated with ZIKV infection of the placenta and fetal brain. We identified ZIKV within trophoblasts of the maternal and fetal placenta, consistent with a trans-placental infection route. Antibody blockade of Ifnar1 signaling in WT pregnant mice enhanced ZIKV trans-placental infection although it did not result in fetal death. These models will facilitate the study of ZIKV pathogenesis, in utero transmission, and testing of therapies and vaccines to prevent congenital malformations.

Dengue Antibodies, then Zika: A Fatal Sequence in Mice.

Anti-Dengue virus (DENV) antibodies can be either protective or pathogenic in humans with prior DENV infection. In a recent issue of Science, Bardina et al. (2017) demonstrated that passive transfer of immune plasma against DENV and West Nile virus (WNV) can enhance Zika virus (ZIKV) infection and pathogenesis in mice.

Regulation of cGAS and STING signaling during inflammation and infection.

Stimulator of interferon genes (STING) is a sensor of cyclic dinucleotides including cyclic GMP-AMP, which is produced by cyclic GMP-AMP synthase (cGAS) in response to cytosolic DNA. The cGAS-STING signaling pathway regulates both innate and adaptive immune responses, as well as fundamental cellular functions such as autophagy, senescence, and apoptosis. Mutations leading to constitutive activation of STING cause devastating human diseases. Thus, the cGAS-STING pathway is of great interest because of its role in diverse cellular processes and because of the potential therapeutic implications of targeting cGAS and STING. Here, we review molecular and cellular mechanisms of STING signaling, and we propose a framework for understanding the immunological and other cellular functions of STING in the context of disease.

Norovirus evolution in immunodeficient mice reveals potentiated pathogenicity via a single nucleotide change in the viral capsid.

Interferons (IFNs) are key controllers of viral replication, with intact IFN responses suppressing virus growth and spread. Using the murine norovirus (MNoV) system, we show that IFNs exert selective pressure to limit the pathogenic evolutionary potential of this enteric virus. In animals lacking type I IFN signaling, the nonlethal MNoV strain CR6 rapidly acquired enhanced virulence via conversion of a single nucleotide. This nucleotide change resulted in amino acid substitution F514I in the viral capsid, which led to >10,000-fold higher replication in systemic organs including the brain. Pathogenicity was mediated by enhanced recruitment and infection of intestinal myeloid cells and increased extraintestinal dissemination of virus. Interestingly, the trade-off for this mutation was reduced fitness in an IFN-competent host, in which CR6 bearing F514I exhibited decreased intestinal replication and shedding. In an immunodeficient context, a spontaneous amino acid change can thus convert a relatively avirulent viral strain into a lethal pathogen.

A CRISPR screen defines a signal peptide processing pathway required by flaviviruses.

Flaviviruses infect hundreds of millions of people annually, and no antiviral therapy is available. We performed a genome-wide CRISPR/Cas9-based screen to identify host genes that, when edited, resulted in reduced flavivirus infection. Here, we validated nine human genes required for flavivirus infectivity, and these were associated with endoplasmic reticulum functions including translocation, protein degradation, and N-linked glycosylation. In particular, a subset of endoplasmic reticulum-associated signal peptidase complex (SPCS) proteins was necessary for proper cleavage of the flavivirus structural proteins (prM and E) and secretion of viral particles. Loss of SPCS1 expression resulted in markedly reduced yield of all Flaviviridae family members tested (West Nile, Dengue, Zika, yellow fever, Japanese encephalitis, and hepatitis C viruses), but had little impact on alphavirus, bunyavirus, or rhabdovirus infection or the surface expression or secretion of diverse host proteins. We found that SPCS1 dependence could be bypassed by replacing the native prM protein leader sequences with a class I major histocompatibility complex (MHC) antigen leader sequence. Thus, SPCS1, either directly or indirectly via its interactions with unknown host proteins, preferentially promotes the processing of specific protein cargo, and Flaviviridae have a unique dependence on this signal peptide processing pathway. SPCS1 and other signal processing pathway members could represent pharmacological targets for inhibiting infection by the expanding number of flaviviruses of medical concern.

Nitro-fatty acids are formed in response to virus infection and are potent inhibitors of STING palmitoylation and signaling.

The adaptor molecule stimulator of IFN genes (STING) is central to production of type I IFNs in response to infection with DNA viruses and to presence of host DNA in the cytosol. Excessive release of type I IFNs through STING-dependent mechanisms has emerged as a central driver of several interferonopathies, including systemic lupus erythematosus (SLE), Aicardi-Goutières syndrome (AGS), and stimulator of IFN genes-associated vasculopathy with onset in infancy (SAVI). The involvement of STING in these diseases points to an unmet need for the development of agents that inhibit STING signaling. Here, we report that endogenously formed nitro-fatty acids can covalently modify STING by nitro-alkylation. These nitro-alkylations inhibit STING palmitoylation, STING signaling, and subsequently, the release of type I IFN in both human and murine cells. Furthermore, treatment with nitro-fatty acids was sufficient to inhibit production of type I IFN in fibroblasts derived from SAVI patients with a gain-of-function mutation in STING. In conclusion, we have identified nitro-fatty acids as endogenously formed inhibitors of STING signaling and propose for these lipids to be considered in the treatment of STING-dependent inflammatory diseases.

A Human STAT1 Gain-of-Function Mutation Impairs CD8+ T Cell Responses against Gammaherpesvirus 68.

Autosomal dominant STAT1 mutations in humans have been associated with chronic mucocutaneous candidiasis (CMC), as well as with increased susceptibility to herpesvirus infections. Prior studies have focused on mucosal and Th17-mediated immunity against Candida, but mechanisms of impaired antiviral immunity have not previously been examined. To begin to explore the mechanisms of STAT1-associated immunodeficiency against herpesviruses, we generated heterozygous STAT1 R274W knock-in mice that have a frequently reported STAT1 mutation associated in humans with susceptibility to herpesvirus infections. In primary macrophages and fibroblasts, we found that STAT1 R274W had no appreciable effect on cell-intrinsic immunity against herpes simplex virus 1 (HSV-1) or gammaherpesvirus 68 (γHV68) infection. However, intraperitoneal inoculation of mice with γHV68 was associated with impaired control of infection at day 14 in STAT1 R274W mice compared with that in wild-type (WT) littermate control animals. Infection of STAT1 R274W mice was associated with paradoxically decreased expression of IFN-stimulated genes (ISGs) and gamma interferon (IFN-γ), likely secondary to defective CD4+ and CD8+ T cell responses, including diminished numbers of antigen-specific CD8+ T cells. Viral pathogenesis studies in WT and STAT1 R274W mixed bone marrow chimeric mice revealed that the presence of WT leukocytes was sufficient to limit infection and that antigen-specific STAT1 R274W CD8+ T cell responses were impaired even in the presence of WT leukocytes. Thus, in addition to regulating Th17 responses against Candida, a STAT1 gain-of-function mutant impedes antigen-specific T cell responses against a common gammaherpesvirus in mice.IMPORTANCE Mechanisms of immunodeficiency related to STAT1 gain of function have not been previously studied in an animal model of viral pathogenesis. Using virological and immunological techniques, we examined the immune response to γHV68 in heterozygous mice that have an autosomal dominant mutation in the STAT1 coiled-coil domain (STAT1 R274W). We observed impaired control of infection, which was associated with diminished production of gamma interferon (IFN-γ), fewer effector CD4+ and CD8+ T cells, and a reduction in the number of antigen-specific CD8+ T cells. These findings indicate that a STAT1 gain-of-function mutation limits production of antiviral T cells, likely contributing to immunodeficiency against herpesviruses.

A Human Gain-of-Function STING Mutation Causes Immunodeficiency and Gammaherpesvirus-Induced Pulmonary Fibrosis in Mice.

We previously generated STING N153S knock-in mice that have a human disease-associated gain-of-function mutation in STING. Patients with this mutation (STING N154S in humans) develop STING-associated vasculopathy with onset in infancy (SAVI), a severe pediatric autoinflammatory disease characterized by pulmonary fibrosis. Since this mutation promotes the upregulation of antiviral type I interferon-stimulated genes (ISGs), we hypothesized that STING N153S knock-in mice may develop more severe autoinflammatory disease in response to a virus challenge. To test this hypothesis, we infected heterozygous STING N153S mice with murine gammaherpesvirus 68 (γHV68). STING N153S mice were highly vulnerable to infection and developed pulmonary fibrosis after infection. In addition to impairing CD8+ T cell responses and humoral immunity, STING N153S also promoted the replication of γHV68 in cultured macrophages. In further support of a combined innate and adaptive immunodeficiency, γHV68 infection was more severe in Rag1-/- STING N153S mice than in Rag1-/- littermate mice, which completely lack adaptive immunity. Thus, a gain-of-function STING mutation creates a combined innate and adaptive immunodeficiency that leads to virus-induced pulmonary fibrosis.IMPORTANCE A variety of human rheumatologic disease-causing mutations have recently been identified. Some of these mutations are found in viral nucleic acid-sensing proteins, but whether viruses can influence the onset or progression of these human diseases is less well understood. One such autoinflammatory disease, called STING-associated vasculopathy with onset in infancy (SAVI), affects children and leads to severe lung disease. We generated mice with a SAVI-associated STING mutation and infected them with γHV68, a common DNA virus that is related to human Epstein-Barr virus. Mice with the human disease-causing STING mutation were more vulnerable to infection than wild-type littermate control animals. Furthermore, the STING mutant mice developed lung fibrosis similar to that of patients with SAVI. These findings reveal that a human STING mutation creates severe immunodeficiency, leading to virus-induced lung disease in mice.

STING-associated lung disease in mice relies on T cells but not type I interferon.

BACKGROUND: Monogenic interferonopathies are thought to be mediated by type I interferon. For example, a gain-of-function mutation in stimulator of interferon genes (STING; N153S) upregulates type I interferon-stimulated genes and causes perivascular inflammatory lung disease in mice. The equivalent mutation in human subjects also causes lung disease, which is thought to require signaling through the cyclic GMP-AMP synthase (cGAS)-STING pathway and subsequent activation of interferon regulatory factors (IRFs) 3 and 7, type I interferon, and interferon-stimulated genes. OBJECTIVE: We set out to define the roles of cGAS, IRF3, IRF7, the type I interferon receptor (IFN-α and IFN-ß receptor subunit 1 [IFNAR1]), T cells, and B cells in spontaneous lung disease in STING N153S mice. METHODS: STING N153S mice were crossed to animals lacking cGAS, IRF3/IRF7, IFNAR1, adaptive immunity, αß T cells, and mature B cells. Mice were evaluated for spontaneous lung disease. Additionally, bone marrow chimeric mice were assessed for lung disease severity and survival. RESULTS: Lung disease in STING N153S mice developed independently of cGAS, IRF3/IRF7, and IFNAR1. Bone marrow transplantation revealed that certain features of STING N153S-associated disease are intrinsic to the hematopoietic compartment. Recombination-activating gene 1 (Rag1)-/- STING N153S mice that lack adaptive immunity had no lung disease, and T-cell receptor ß chain (Tcrb)-/- STING N153S animals only had mild disease. STING N153S led to a reduction in percentages and numbers of naive and regulatory T cells, as well as an increased frequency of cytokine-producing effector T cells. CONCLUSION: Spontaneous lung disease in STING N153S mice develops independently of type I interferon signaling and cGAS. STING N153S relies primarily on T cells to promote lung disease in mice.

HOIL1 Is Essential for the Induction of Type I and III Interferons by MDA5 and Regulates Persistent Murine Norovirus Infection.

The linear ubiquitin chain assembly complex (LUBAC), composed of heme-oxidized IRP2 ubiquitin ligase 1 (HOIL1), HOIL1-interacting protein (HOIP), and SHANK-associated RH domain-interacting protein (SHARPIN), is a crucial regulator of multiple immune signaling pathways. In humans, HOIL1 or HOIP deficiency is associated with an immune disorder involving autoinflammation, immunodeficiency, and inflammatory bowel disease (IBD)-like symptoms. During viral infection, LUBAC is reported to inhibit the induction of interferon (IFN) by the cytosolic RNA sensor retinoic acid-inducible gene I (RIG-I). Surprisingly, we found that HOIL1 is essential for the induction of both type I and type III IFNs, as well as the phosphorylation of IFN regulatory factor 3 (IRF3), during murine norovirus (MNoV) infection in cultured dendritic cells. The RIG-I-like receptor, melanoma differentiation-associated protein 5 (MDA5), is also required for IFN induction and IRF3 phosphorylation during MNoV infection. Furthermore, HOIL1 and MDA5 were required for IFN induction after Theiler's murine encephalomyelitis virus infection and poly(I·C) transfection, but not Sendai virus or vesicular stomatitis virus infection, indicating that HOIL1 and LUBAC are required selectively for MDA5 signaling. Moreover, Hoil1-/- mice exhibited defective control of acute and persistent murine norovirus infection and defective regulation of MNoV persistence by the microbiome as also observed previously for mice deficient in interferon lambda (IFN-λ) receptor, signal transducer and activator of transcription factor 1 (STAT1), and IRF3. These data indicate that LUBAC plays a critical role in IFN induction to control RNA viruses sensed by MDA5.IMPORTANCE Human noroviruses are a leading cause of gastroenteritis throughout the world but are challenging to study in vivo and in vitro Murine norovirus (MNoV) provides a tractable genetic and small-animal model to study norovirus biology and immune responses. Interferons are critical mediators of antiviral immunity, but excessive expression can dysregulate the immune system. IFN-λ plays an important role at mucosal surfaces, including the gastrointestinal tract, and both IFN-λ and commensal enteric bacteria are important modulators of persistent MNoV infection. LUBAC, of which HOIL1 is a component, is reported to inhibit type I IFN induction after RIG-I stimulation. We show, in contrast, that HOIL1 is critical for type I and III IFN induction during infection with MNoV, a virus that preferentially activates MDA5. Moreover, HOIL1 regulates MNoV infection in vivo These data reveal distinct functions for LUBAC in these closely related signaling pathways and in modulation of IFN expression.

Interferon regulatory factor 5-dependent immune responses in the draining lymph node protect against West Nile virus infection.

UNLABELLED: Upon activation of Toll-like and RIG-I-like receptor signaling pathways, the transcription factor IRF5 translocates to the nucleus and induces antiviral immune programs. The recent discovery of a homozygous mutation in the immunoregulatory gene guanine exchange factor dedicator of cytokinesis 2 (Dock2mu/mu) in several Irf5-/- mouse colonies has complicated interpretation of immune functions previously ascribed to IRF5. To define the antiviral functions of IRF5 in vivo, we infected backcrossed Irf5-/-×Dock2wt/wt mice (here called Irf5-/- mice) and independently generated CMV-Cre Irf5fl/fl mice with West Nile virus (WNV), a pathogenic neurotropic flavivirus. Compared to congenic wild-type animals, Irf5-/- and CMV-Cre Irf5fl/fl mice were more vulnerable to WNV infection, and this phenotype was associated with increased infection in peripheral organs, which resulted in higher virus titers in the central nervous system. The loss of IRF5, however, was associated with only small differences in the type I interferon response systemically and in the draining lymph node during WNV infection. Instead, lower levels of several other proinflammatory cytokines and chemokines, as well as fewer and less activated immune cells, were detected in the draining lymph node 2 days after WNV infection. WNV-specific antibody responses in Irf5-/- mice also were blunted in the context of live or inactivated virus infection and this was associated with fewer antigen-specific memory B cells and long-lived plasma cells. Our results with Irf5-/- mice establish a key role for IRF5 in shaping the early innate immune response in the draining lymph node, which impacts the spread of virus infection, optimal B cell immunity, and disease pathogenesis. IMPORTANCE: Although the roles of IRF3 and IRF7 in orchestrating innate and adaptive immunity after viral infection are established, the function of the related transcription factor IRF5 remains less certain. Prior studies in Irf5-/- mice reported conflicting results as to the contribution of IRF5 in regulating type I interferon and adaptive immune responses. The lack of clarity may stem from a recently discovered homozygous loss-of-function mutation of the immunoregulatory gene Dock2 in several colonies of Irf5-/- mice. Here, using a mouse model with a deficiency in IRF5 and wild-type Dock2 alleles, we investigated how IRF5 modulates West Nile virus (WNV) pathogenesis and host immune responses. Our in vivo studies indicate that IRF5 has a key role in shaping the early proinflammatory cytokine response in the draining lymph node, which impacts immunity and control of WNV infection.

Inherited C-terminal TREX1 variants disrupt homology-directed repair to cause senescence and DNA damage phenotypes in Drosophila, mice, and humans.

Age-related microangiopathy, also known as small vessel disease (SVD), causes damage to the brain, retina, liver, and kidney. Based on the DNA damage theory of aging, we reasoned that genomic instability may underlie an SVD caused by dominant C-terminal variants in TREX1, the most abundant 3'-5' DNA exonuclease in mammals. C-terminal TREX1 variants cause an adult-onset SVD known as retinal vasculopathy with cerebral leukoencephalopathy (RVCL or RVCL-S). In RVCL, an aberrant, C-terminally truncated TREX1 mislocalizes to the nucleus due to deletion of its ER-anchoring domain. Since RVCL pathology mimics that of radiation injury, we reasoned that nuclear TREX1 would cause DNA damage. Here, we show that RVCL-associated TREX1 variants trigger DNA damage in humans, mice, and Drosophila, and that cells expressing RVCL mutant TREX1 are more vulnerable to DNA damage induced by chemotherapy and cytokines that up-regulate TREX1, leading to depletion of TREX1-high cells in RVCL mice. RVCL-associated TREX1 mutants inhibit homology-directed repair (HDR), causing DNA deletions and vulnerablility to PARP inhibitors. In women with RVCL, we observe early-onset breast cancer, similar to patients with BRCA1/2 variants. Our results provide a mechanistic basis linking aberrant TREX1 activity to the DNA damage theory of aging, premature senescence, and microvascular disease.

Core 1-derived O-glycans are essential E-selectin ligands on neutrophils.

Neutrophils roll on E-selectin in inflamed venules through interactions with cell-surface glycoconjugates. The identification of physiologic E-selectin ligands on neutrophils has been elusive. Current evidence suggests that P-selectin glycoprotein ligand-1 (PSGL-1), E-selectin ligand-1 (ESL-1), and CD44 encompass all glycoprotein ligands for E-selectin; that ESL-1 and CD44 use N-glycans to bind to E-selectin; and that neutrophils lacking core 2 O-glycans have partially defective interactions with E-selectin. These data imply that N-glycans on ESL-1 and CD44 and O-glycans on PSGL-1 constitute all E-selectin ligands, with neither glycan subset having a dominant role. The enzyme T-synthase transfers Gal to GalNAcalpha1-Ser/Thr to form the core 1 structure Galbeta1-3GalNAcalpha1-Ser/Thr, a precursor for core 2 and extended core 1 O-glycans that might serve as selectin ligands. Here, using mice lacking T-synthase in endothelial and hematopoietic cells, we found that E-selectin bound to CD44 and ESL-1 in lysates of T-synthase-deficient neutrophils. However, the cells exhibited markedly impaired rolling on E-selectin in vitro and in vivo, failed to activate beta2 integrins while rolling, and did not emigrate into inflamed tissues. These defects were more severe than those of neutrophils lacking PSGL-1, CD44, and the mucin CD43. Our results demonstrate that core 1-derived O-glycans are essential E-selectin ligands; that some of these O-glycans are on protein(s) other than PSGL-1, CD44, and CD43; and that PSGL-1, CD44, and ESL-1 do not constitute all glycoprotein ligands for E-selectin.

A path towards personalized medicine for autoinflammatory and related diseases.

The human genome project led to the advancement of genetic technologies and genomic medicine for a variety of human diseases, including monogenic autoimmune and autoinflammatory diseases. As a result, the genome of an individual can now be rapidly sequenced at a low cost, and this technology is beginning to change the practice of rheumatology. In this Perspective, we describe how new sequencing technologies combined with careful clinical phenotyping have led to the discovery of rare rheumatic diseases and their corresponding disease-causing mutations. Additionally, we explore ways in which single-gene mutations, including somatic mutations, are creating opportunities to develop personalized medicines. To illustrate this idea, we focus on diseases affecting the TREX1-cGAS-STING pathway, which is associated with monogenic autoinflammatory diseases and vasculopathies. For many of the affected patients and families, there is an urgent, unmet need for the development of personalized therapies. New innovations related to small molecular inhibitors and gene therapies have the potential to benefit these families, and might help drive further innovations that could prove useful for patients with more common forms of autoimmunity and autoinflammation.

Liver pathology in retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations: vasculopathic disease beyond nodular regenerative hyperplasia.

Retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations (RVCL-S) is a rare autosomal dominant disease resulting from a frame-shift mutation in TREX1, an intracellular 3'-5' exonuclease 1. Hepatic findings include an elevated alkaline phosphatase (ALP) and nodular regenerative hyperplasia (NRH). Affected individuals typically succumb to brain lesions before clinically apparent hepatic manifestations; thus, little else is known about the hepatic pathology. Autopsy reports and a liver section from each (n = 11) of three unrelated kindreds with the most common mutation in TREX1 (V235Gfs∗6) were studied with standard and immunohistochemical stains. Cases were compared with "normal liver" controls from similar autopsy years. Cases consisted of six men and five women who died at a median age of 50 yr (range, 41-60 yr.). Seven had elevated ALP. Two had liver atrophy. Foci of NRH were variably detected in all. Inhomogeneous distribution of other findings included patternless parenchymal fibrous bands, approximation of vascular structures, and commonly, architectural changes of vascular structures. Only bile duct epithelia were unaffected. In addition, small trichrome-positive nodules were found along vein walls or isolated in the parenchyma. Rare foci of non-NRH hepatocytic nodules were noted in 3. Increased CD34 and altered α-SMA IHC expression were variably noted. Periportal ductules and perivenular K7 IHC expression were increased to unpredictable degrees. The extensive but inhomogeneous histopathologic findings in livers of autopsied patients with RVCL-S appear to involve hepatic vascular structures. These findings validate inclusion of vascular liver involvement beyond NRH in this complex hereditary disorder.

Cytoplasmic domain of P-selectin glycoprotein ligand-1 facilitates dimerization and export from the endoplasmic reticulum.

P-selectin glycoprotein ligand-1 (PSGL-1) is a homodimeric transmembrane mucin on leukocytes. During inflammation, reversible interactions of PSGL-1 with selectins mediate leukocyte rolling on vascular surfaces. The transmembrane domain of PSGL-1 is required for dimerization, and the cytoplasmic domain propagates signals that activate ß(2) integrins to slow rolling on integrin ligands. Leukocytes from knock-in "ΔCD" mice express a truncated PSGL-1 that lacks the cytoplasmic domain. Unexpectedly, they have 10-fold less PSGL-1 on their surfaces than WT leukocytes. Using glycosidases, proteases, Western blotting, confocal microscopy, cell-surface cross-linking, FRET, and pulse-chase metabolic labeling, we demonstrate that deleting the cytoplasmic domain impaired dimerization and delayed export of PSGL-1 from the endoplasmic reticulum (ER), markedly increasing a monomeric precursor in the ER and decreasing mature PSGL-1 on the cell surface. A monomeric full-length PSGL-1 made by substituting the transmembrane domain with that of CD43 exited the ER normally, revealing that dimerization was not required for ER export. Thus, the transmembrane and cytoplasmic domains cooperate to promote dimerization of PSGL-1. Furthermore, the cytoplasmic domain provides a key signal to export precursors of PSGL-1 from the ER to the Golgi apparatus en route to the cell surface.

E-selectin engages PSGL-1 and CD44 through a common signaling pathway to induce integrin alphaLbeta2-mediated slow leukocyte rolling.

In inflamed venules, neutrophils rolling on E-selectin induce integrin alpha(L)beta(2)-dependent slow rolling on intercellular adhesion molecule-1 by activating Src family kinases (SFKs), DAP12 and Fc receptor-gamma (FcRgamma), spleen tyrosine kinase (Syk), and p38. E-selectin signaling cooperates with chemokine signaling to recruit neutrophils into tissues. Previous studies identified P-selectin glycoprotein ligand-1 (PSGL-1) as the essential E-selectin ligand and Fgr as the only SFK that initiate signaling to slow rolling. In contrast, we found that E-selectin engagement of PSGL-1 or CD44 triggered slow rolling through a common, lipid raft-dependent pathway that used the SFKs Hck and Lyn as well as Fgr. We identified the Tec kinase Bruton tyrosine kinase as a key signaling intermediate between Syk and p38. E-selectin engagement of PSGL-1 was dependent on its cytoplasmic domain to activate SFKs and slow rolling. Although recruiting phosphoinositide-3-kinase to the PSGL-1 cytoplasmic domain was reported to activate integrins, E-selectin-mediated slow rolling did not require phosphoinositide-3-kinase. Studies in mice confirmed the physiologic significance of these events for neutrophil slow rolling and recruitment during inflammation. Thus, E-selectin triggers common signals through distinct neutrophil glycoproteins to induce alpha(L)beta(2)-dependent slow rolling.

The IFN-γ receptor promotes immune dysregulation and disease in STING gain-of-function mice.

STING gain-of-function mutations cause STING-associated vasculopathy with onset in infancy (SAVI) in humans, a disease characterized by spontaneous lung inflammation and fibrosis. Mice with STING gain-of-function mutations (SAVI mice) develop αß T cell-dependent lung disease and also lack lymph nodes. Although SAVI has been regarded as a type I interferonopathy, the relative contributions of the three interferon receptors are incompletely understood. Here, we show that STING gain of function led to upregulation of IFN-γ-induced chemokines in the lungs of SAVI mice and that deletion of the type II IFN receptor (IFNGR1), but not the type I IFN receptor (IFNAR1) or type III IFN receptor (IFNλR1), ameliorated lung disease and restored lymph node development in SAVI mice. Furthermore, deletion of IFNGR1, but not IFNAR1 or IFNλR1, corrected the ratio of effector to Tregs in SAVI mice and in mixed bone marrow chimeric mice. Finally, cultured SAVI mouse macrophages were hyperresponsive to IFN-γ, but not IFN-ß, in terms of Cxcl9 upregulation and cell activation. These results demonstrate that IFNGR1 plays a major role in autoinflammation and immune dysregulation mediated by STING gain of function.

Platelet glycoprotein Ibalpha forms catch bonds with human WT vWF but not with type 2B von Willebrand disease vWF.

Arterial blood flow enhances glycoprotein Ibalpha (GPIbalpha) binding to vWF, which initiates platelet adhesion to injured vessels. Mutations in the vWF A1 domain that cause type 2B von Willebrand disease (vWD) reduce the flow requirement for adhesion. Here we show that increasing force on GPIbalpha/vWF bonds first prolonged ("catch") and then shortened ("slip") bond lifetimes. Two type 2B vWD A1 domain mutants, R1306Q and R1450E, converted catch bonds to slip bonds by prolonging bond lifetimes at low forces. Steered molecular dynamics simulations of GPIbalpha dissociating from the A1 domain suggested mechanisms for catch bonds and their conversion by the A1 domain mutations. Catch bonds caused platelets and GPIbalpha-coated microspheres to roll more slowly on WT vWF and WT A1 domains as flow increased from suboptimal levels, explaining flow-enhanced rolling. Longer bond lifetimes at low forces eliminated the flow requirement for rolling on R1306Q and R1450E mutant A1 domains. Flowing platelets agglutinated with microspheres bearing R1306Q or R1450E mutant A1 domains, but not WT A1 domains. Therefore, catch bonds may prevent vWF multimers from agglutinating platelets. A disintegrin and metalloproteinase with a thrombospondin type 1 motif-13 (ADAMTS-13) reduced platelet agglutination with microspheres bearing a tridomain A1A2A3 vWF fragment with the R1450E mutation in a shear-dependent manner. We conclude that in type 2B vWD, prolonged lifetimes of vWF bonds with GPIbalpha on circulating platelets may allow ADAMTS-13 to deplete large vWF multimers, causing bleeding.

Transferrable protection by gut microbes against STING-associated lung disease.

STING modulates immunity by responding to bacterial and endogenous cyclic dinucleotides (CDNs). Humans and mice with STING gain-of-function mutations develop a syndrome known as STING-associated vasculopathy with onset in infancy (SAVI), which is characterized by inflammatory or fibrosing lung disease. We hypothesized that hyperresponsiveness of gain-of-function STING to bacterial CDNs might explain autoinflammatory lung disease in SAVI mice. We report that depletion of gut microbes with oral antibiotics (vancomycin, neomycin, and ampicillin [VNA]) nearly eliminates lung disease in SAVI mice, implying that gut microbes might promote STING-associated autoinflammation. However, we show that germ-free SAVI mice still develop severe autoinflammatory disease and that transferring gut microbiota from antibiotics-treated mice to germ-free animals eliminates lung inflammation. Depletion of anaerobes with metronidazole abolishes the protective effect of the VNA antibiotics cocktail, and recolonization with the metronidazole-sensitive anaerobe Bacteroides thetaiotaomicron prevents disease, confirming a protective role of a metronidazole-sensitive microbe in a model of SAVI.